Death of hair cells is the leading cause of hearing and balance disorders, which impact the lives and health of millions of people in the United States. In adult mammals, auditory hair cells are not replaced, but some hair cell regeneration may occur in the utricle, a balance organ. The long-term goal of our research is to develop therapies for hearing and balance deficits in humans. Toward this goal, we have studied adult mouse utricles to characterize their innate capacity for hair cell regeneration and to identify the cellular and molecular factors that block resident progenitors (supporting cells) from forming new hair cells after damage. Here, we propose to extend these studies into other regions of the inner ear, including the cochlea. We employed a new transgenic mouse (Pou4f3DTR) in which selective hair cell loss can be induced by administration of diphtheria toxin (DT). In utricles, 96% of hair cells were killed by DT treatment, and 17% of hair cells were replaced, apparently by a non-mitotic process. Most new hair cells were located in and around the striola. Surprisingly, supporting cells throughout the utricle upregulated Atoh1 after damage, suggesting widespread initiation of transdifferentiation into hair cells. These findings indicate that unidentified factors prevent many activated supporting cells from forming replacement hair cells. Our studies suggest notch signaling is one factor blocking both the widespread initial upregulation of Atoh1 and the regional transdifferentiation of supporting cells into hair cells. Exciting novel observations suggest cell death is another factor preventing hair cell replacement, since transdifferentiating supporting cells in extrastriolar regions appear to undergo apoptosis and phagocytosis before they can develop properties of mature hair cells. We hypothesize that the mechanisms blocking supporting cells from transdifferentiating into hair cells in mouse utricles are fundamental inhibitors of hair cell replacement in all mammalian inner ear epithelia, including the auditory organ. In adult mice, we propose to examine how notch signaling and apoptosis curtail hair cell regeneration in the utricle, as well as in the lateral ampulla and the cochlea, after damage. In Aim 1, loss-of-function and gain-of-function experiments will test if notch activity specifically blocks initiation and progression of supporting cell transdifferentiation and survival of hair cell precursors after damage. In Aim 2, we will assess if supporting cells initiate hair cell replacement in damaged inner ear organs in vivo and determine how many supporting cells convert into bona fide hair cells without dividing. In Aim 3, we will determine if activated supporting cells in damaged inner ear organs undergo apoptosis in vivo and if inhibition of this process in utricles increases hair cell replacement. These experiments will establish definitively whether supporting cells in adult mice directly transdifferentiate into hair cells and will reveal cellular and molecular obstacles to hair cell regeneration in mammals that may be key targets for future hearing restoration therapy.